Method for stimulating wound healing

ABSTRACT

The present invention relates to a method for stimulating wound healing, and more particularly to a method for stimulating wound healing in a subject in need thereof, comprising administering to a wound of the subject an effective amount for stimulating wound healing of a composition, wherein the composition comprises p43 having an amino acid sequence set forth in SEQ ID NO: 1 or functional equivalents thereof. The composition used in the method of the present invention can be efficiently utilized for the wound healing, since p43, an effective ingredient of the composition, has an excellent effect on the wound healing by its action including the induction of macrophage/monocyte and endothelial cell re-epithelization, proliferation of fibroblasts or angiogenesis.

This application claims the priority of Korea Patent Application No.2002-42858, filed Jul. 22, 2002.

FIELD OF THE INVENTION

The present invention relates to a method for stimulating wound healing,and more particularly to a method for stimulating wound healing in asubject in need thereof, comprising: administering to a wound of thesubject an effective amount for stimulating wound healing of acomposition, wherein the composition comprising p43 having an amino acidsequence set forth in SEQ ID NO: 1 or functional equivalents thereof.

BACKGROUND OF THE INVENTION

In general, wound healing is any reaction of tissue against damageinflicted upon the tissue, and it is known as a complex biologicalprocess comprising chemotaxis, cell differentiation and replication,synthesis of matrix protein, angiogenesis and reconstitution of wound,as a series of tissue repair processes (Steed D. L. et al., Clin. Plast.Surg. 25:397, 1998). As one of the typical factors for controlling woundhealing process, the growth factor may be considered, which regulatecell growth, cell differentiation, cell metabolism and the like throughthe general wound healing process, as well as the environment around awound, and have consistently been used for the development of treatmentagents. In addition, various kinds of cytokine are known to participatein wound healing. As an example of the cytokine, there is transforminggrowth factor-β (TGF-β). The TGF-β is involved in the growth anddifferentiation of various cells. It has been reported that the TGF-β isinvolved in several complicated functions including growth control, theregulation of immune response, the stimulation of osteogenesis, theinduction of cartilage-specific macromolecule and the promotion of woundhealing (Bennett, N. T. et al., Am. J. Surg. 165:728, 1993).

According to whether skin deficiency exists or not, a wound may beclassified into two types and may be treated with different healingmechanisms. When a wound does not involve skin deficiency, as in mostmild injuries, for example a case of having injury only to theepidermis, keratinocytes move from the wound periphery, cover the wound,and reform new epidermis and keratin (Knighton, D. R. and Fiegel, V. D.,Invest. Radiol. 26:604-611, 1991). However, when entire skin layers aretotally injured or destroyed, new connective tissues, called granulationtissues, fill wound space where regenerated epidermis from the peripheryof the wound sites must be covered. The granulation tissues are formedby the deposition of extracellular matrix components, such as collagen,from fibroblasts moving into the wound space. Consequently, the healingmechanisms of wound significantly varied depending on the presence ofsuch skin deficiency. Successful wound healing requires completing aseries of wound healing multistage processes. Since the deletion of oneor more of wound healing-related components prevents the wound healingand skin repair, the wound remains exposed. Such an exposed wound can beeasily infected, resulting in delayed healing processes, and theformation of ulcers and erosion of skin. There is a need for thedevelopment of a drug promoting the growth of granulation tissue and theregeneration of skin on wound involving skin deficiency.

Meanwhile, p43 is a protein consisting of 312 amino acids which isassociated with a multi-tRNA synthetase complex to facilitate thecatalytic activity of the bound enzyme. The p43 is highly expressed bymicroneuron in the lesions of autoimmune diseases in vitro includingencephalomyelitis, neuritis and uveitis. The phenomenon in which p43 ishighly expressed at certain developmental stages and tissues suggeststhat p43 is related to inflammation response and cell apoptosis (Berger,A. C. et al., J. Immunother. 23:519-527, 2000). We have previously shownthat p43 can be employed as an effective cytokine and an anti-tumoragent (PCT International application No. PCT/KR00/00630). However, it isstill not shown that p43 may be employed for treating wound. Weidentified that p43 has a novel activity of promoting wound healingprocess by acting on the wound healing process comprising theinflammation stage, epithelization stage and angiogenesis stage, andcompleted the present invention.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a methodfor stimulating wound healing in a subject in need thereof, comprisingadministering to a wound of the subject an effective amount forstimulating wound healing of a composition, wherein the compositioncomprises p43 having an amino acid sequence set forth in SEQ ID NO: 1 orfunctional equivalents thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of the presentinvention will become more apparent from the following detaileddescription when taken in conjunction with the accompanying drawings inwhich:

FIG. 1 is a schematic drawing showing where the gene trap is insertedwithin p43 mutant allele, wherein A, K, S and H indicate the restrictionsites for Apa I, Kpn I, Sac I and HindIII, respectively. IF3, IR3 andpKOF2 are primers for PCR.

FIG. 2 shows the results of Southern blot (A) and PCR (B) foridentifying the mutation of p43.

+/+: wild type mouse (WT);

+/−: heterozygous mutant mouse;

−/−: homozygous mutant mouse (Ho).

FIG. 3 shows the results of Northern blot (A) and Western blot fordetermining the expression of p43.

+/+: wild type mouse (WT);

+/−: heterozygous mutant mouse;

−/−: homozygous mutant mouse (Ho).

FIG. 4 is a photograph showing wound healing levels with the lapse oftime (A) and a bar graph representing a percentage of wound healinglevels to an initial wound area.

Ho: homozygous mutant mouse;

WT: wild type mouse which is not treated with p43 (control);

WT+p43: wild type mouse treated with p43.

FIG. 5 shows the result of histochemical analysis representing theeffect of p43 to stimulate wound healing.

Arrows: advancing epithelial layers;

Purple region: granulation tissues.

FIG. 6 shows the results of immunostaining for determining thegeneration levels of macrophage/monocyte and endothelial cells.

FIG. 7 is a bar graph representing the effect of p43 on theproliferation of fibroblasts.

FIG. 8 shows the result of histochemical analysis representing theeffect of p43 on the proliferation of fibroblasts.

Left: hematoxylin and eosin staining result of the cross-section of thewound area (arrows: the boundary of the wound area);

Right: immuno-fluorescence staining results of the boxed region in thewound area with the antibody against Ki-67.

FIG. 9 shows the results of wound migration assay (A), tube formationassay (B) and CAM assay (C) for estimating the effect of p43 in bloodvessel generation.

FIG. 10 shows the results of the matrigel plug assay for confirming thestimulation of neovascularization by p43.

FIG. 11 is a bar graph representing the hemoglobin (Hb) contents withinmatrigel containing p43.

DETAILED DESCRIPTION OF THE INVENTION

According to an aspect of the present invention, there is provided amethod for stimulating wound healing in a subject in need thereof,comprising administering to a wound of the subject an amount of acomposition effective for stimulating wound healing, wherein thecomposition comprises a polypeptide selected from the group consistingof:

(a) a polypeptide having an amino acid sequence set forth in SEQ ID NO:1; and

(b) a polypeptide having at least 70% sequence homology with thepolypeptide of (a).

Hereinafter, the term “wound” includes any injury to any portion of thebody of a subject including, but not limited to, acute conditions suchas thermal burns, chemical burns, radiation burns, burns caused byexcess exposure to ultraviolet radiation such as sunburn, damage tobodily tissues such as the perineum as a result of labor and childbirth,including injuries sustained during medical procedures such asepisiotomies, trauma-induced injuries including cuts, incisions,excoriations, those injuries sustained in automobile and othermechanical accidents, and those caused by bullets, knives and otherweapons, ulcer such as pressure ulcer, plaster ulcer and decubitusulcer, and post-surgical injuries, as well as chronic conditions such aspressure sores, bedsores, conditions related to diabetes and poorcirculation, and all types of acne. In addition, said wound includesdermatitis such as impetigo, intertrigo, folliculitis and eczema.

Areas of the body which can be treated with the present inventioninclude, but are not limited to, skin, muscle and internal organs.Hereinafter, the term “subject” refers to a human or lower animal onwhom the present invention is practiced.

The composition used in the present invention comprises, as an effectiveingredient, p43 having an amino acid sequence set forth in SEQ ID NO: 1.The composition used in the present invention may also comprise thefunctional equivalents of the p43 as well as the p43. “Functionalequivalents”, as used herein, refer to polypeptides having thephysiological activity to stimulate wound healing substantiallyequivalent with that of p43 having the amino acid sequences set forth inSEQ ID NO: 1. The functional equivalents may be a polypeptide having atleast 70% sequence homology, preferably at least 80%, more preferably atleast 90%, and most preferably at least 95%, compared to the amino acidsequence set forth in SEQ ID NO: 1. The functional equivalents includeamino acid sequence variants, for example, in which a portion or all ofnatural p43 amino acid sequence is substituted, or a portion of the p43amino acid sequence is deleted or added. The substitution of the aminoacid is preferably a conservative substitution. For example, the aminoacids occurring the conservative substitution in nature includealiphatic amino acids (Gly, Ala, Pro); hydrophobic amino acids (Ile,Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids(Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn), andsulfur-containing amino acids (Cys, Met). The amino acid deletion ispreferably located at a portion that does not affect the physiologicalactivity of p43.

Herein, p43 or functional equivalents thereof can be administered (orapplied) in an appropriate amount depending on the size of wound. In thecase of a circular excision wound of bilateral symmetry having adiameter of 0.5 cm, the p43 is preferably administered in a amount rangeof about 1 ng to 5 μg.

The composition comprising, as an effective component, the p43 orfunctional equivalents thereof according to the present invention mayfurther comprise at least one selected from the group consisting ofantibiotics, such as tetracycline, oxytetracycline, gentamicin, neomycinsulfate, bacitracin and polymyxin B sulfate; antihistamines, such asdiphenhydramine, promethazine, tripelennamine, phenothiazine,chlorophenylamine, antazoline and pantholin; anti-inflammatory drugs;anti-viral drugs; anti-fungal agents; and growth factors, such as PDGF(platelet-derived growth factor), PDAF, PDEGF, TGF-β (transforminggrowth factor-β), PF-4, α FGF, bFGF (basic fibroblast growth factor),VEGF (vascular endothelial growth factor), (3′-1 (growth hormone), EGF(epidermal growth factor) and IGF (insulin-like growth factor).

The composition used in the present invention may be compounded with apharmaceutically acceptable carrier and employed in the form of powder,liniment, gel, lotion, cream, ointment, paste, puff, aerosol orsuppository, and in particular, ointment and paste are more preferable.The carrier may comprise hydrocarbons including petrolatum, liquidparaffin and gelling hydrocarbon; animal or vegetable oils includingmedium chain triglyceride, pig fat, hard fat and cacao oil; higher fattyalcohols including cetanol and stearyl alcohol; fatty acids and estersthereof including stearic acid isopropyl palmitate; aqueous basesincluding polyethylene glycol, 1,3-butyleneglycol, glycerol, gelatin,white sugar and sugar alcohol; emulsifiers including glycerine fattyacid esters, polyoxyl stearate and polyoxyethylene hydrogenated castoroil; conglutinants including acrylate ester and sodium alginate;propellants including liquefied petroleum gas and carbon dioxide; andpreservatives including paraoxybenzoates, depending on each formulationtype.

When damaged skin was exposed to exterior environment, the damagedregion may be infiltrated by toxins or bacteria, resulting ininflammation due to body's defense function. In treatment, the rapidgeneration of granulation tissue should be fostered in the damagedregion and, above all, it is important for bacterial infection orexcessive inflammation to be suppressed. Otherwise, not only much timeis necessary in wound healing but also big scars will remain. The p43having an amino acid sequence set forth in SEQ ID NO: 1 shows excellenteffects on rapid healing of a wound by increasing granulation tissue andstimulating angiogenesis (blood vessel generation) in any woundincluding scald and ulcer regions.

The stimulation of wound healing by p43 identified in the presentinvention is considered to be a result from its multiple effect oninflammation as well as angiogenesis. The attraction ofmacrophage/monocyte by p43 is consistent with our expectation becausevarious proinflammatory cytokines and chemokines such as monocytechemotactic protein 1 (MCP-1) or macrophage inflammatory protein 1α(MIP-1α) have been reported to be induced by p43 [Young-Gyu Ko, et al.,J. Biol. Chem. 276:23028-23033, 2001]. However, the pro-angiogenicactivity of p43 shown in the present invention is surprising because p43was previously shown to suppress the cell proliferation and induceapoptosis of endothelial cells at high concentration [Sun Young Chang,et al., J. Biol. Chem., 277:8388-8394, 2002]. Based on these results,the present invention provides the first evidences on a novel activityof p43 in angiogenesis associated with wound repair.

As can be seen from the foregoing, p43, an effective ingredient of thecomposition of the present invention has a novel physiological activityincluding the induction of macrophage/monocyte and endothelial cell,re-epithelization, proliferation of fibroblasts or angiogenesis.Therefore, it is expected that the method for stimulating wound healingusing the composition containing the p43 or functional equivalentsthereof according to the present invention can be extensively used inthe general medical areas involving wound healing.

In a preferred embodiment of the present invention, the presentinventors introduced a mutation to the structural gene of p43 in mouse.The mutation of the mouse genomic DNA may be effected according toconventional methods used in the prior art, including a gene trapmethod, a gene targeting method or an N-ethyl-N-nitrosourea (ENU)treatment method. Specifically, the gene trap vector was used herein tomutate the mouse genomic DNA. The gene trap vector may be one of thoseconventionally used in the prior art. More preferably, the gene trapvector may be VICTR20 vector. The VICTR20 comprises two functionalunits, a neomycin resistant gene (β-geo/neo^(r)) and a puromycinresistant gene (puro) (see FIG. 1). The neomycin resistant gene isexpressed by an upstream promoter of genomic DNA in which the vector isinserted and the puromycin resistant gene is expressed by thephosphoglycerate kinase-1 (PGK) promoter of the vector. As shown in FIG.1, the gene trap vector, VICTR20 includes long terminal repeats (LTRs)in both ends, a splice accepter (SA), a neomycin resistant gene(neo^(r)), a poly-A sequence (pA), a PGK promoter, a puromycin resistantgene (puro) and a splice donor (SD).

Then, to construct a mutant library, the genomic DNA in which a mutationis derived from the insertion of the gene trap is introduced intoembryonic stem cells of mice. In the mutant library, the clonecontaining the disrupted p43 gene is identified and used to prepareheterozygous mutant mice.

In another preferred embodiment of the present invention, the nucleotidesequence of p43 allele in the p43 mutant mice is determined, and therebyit can be confirmed that the gene trap vector is inserted at the firstintron of the p43 gene (See FIG. 1). The mutation of p43 is determinedby Southern blot and PCR analysis (See FIG. 2) and the mutational effecton the expression of p43 is determined by Northern and Western blot (SeeFIG. 3). The mating of the heterozygous mutant mice generated 16 of wildtype, 53 of heterozygous mutant mice and 28 of homozygous mutant mice.This ratio is close to the Mendelian segregation, indicating nosignificant embryonic lethality of the mutant mice. Interestingly, thehomozygous mutants significantly reduced body size compared to the wildtype mice (data not shown).

It has been reported that p43 is associated with immune mechanism andworks on endothelial cells [Chang S. Y., et al., J. Biol. Chem.,277:8388-8394, 2002; Park H—Y., et al., J. Leukocyte Biol. 71:223-230,2002; Ko Y-G., et al., J. Biol. Chem. 276:23028-23033, 2001]. Weexpected that p43 might be involved in the processes of inflammation andangiogenesis occurring in the wound healing process. Thus, to confirmour expectation, the biopsies were introduced to the wild type andhomozygous mutant mice, and the healing process was compared by grossexamination and histological inspection. The wounds in the mutant mice(Ho) recovered at much slower rate compared to those in the wild typemice and direct administration of the purified p43 to the wound regionof the wild type mice stimulated the healing process (See FIG. 4). Thecross-sections of the wound showed the increase of granulation tissueand rapid re-epithelization in the wounds of the wild type mice treatedwith p43 (See FIG. 5).

Further, immunostaining of the wound regions with an antibody that isspecific to macrophage/monocyte showed the enrichment of these cells inthe wound region tissue of the p43-treated wild type mice (WT+p43) whilefewer numbers of these cells were observed in the wound region tissue ofthe p43 mutant mice (Ho) (See FIG. 6). The macrophage/monocyte-specificantibody may include, but not limited to, anti-MOMA-2 antibody. Theimmunostaining of the wound regions with an antibody specific toendothelial cells showed that vascularization was also significantlyenhanced in the p43-treated wild type mice (WT+p43) while the reversewas the case in the p43 mutants (Ho) (See FIG. 6). The antibody specificto endothelial cell may include, but not limited to, anti-CD31 antibody.

To determine the effect of p43 on the proliferation of different cellsin the wound region, we have treated the cultivated foreskin fibroblastcells with p43. The proliferation of fibroblasts was significantlyincreased in dose-dependent manner by p43 (See FIG. 7). p43 did notstimulate the proliferation of keratinocyte and endothelial cells (datanot shown). To see whether p43 can also show the proliferative effect onthe fibroblasts in the dermal region of the wound area, we havegenerated the wound in the back skin of the mice and treated p43 afterwounding twice a day in every other day. The proliferation of dermalfibroblasts was then compared by the immuno-fluorescence staining withanti-Ki67 antibody 3 days after wounding. The proliferation of dermalfibroblasts was stimulated in the p43 treated wound region (See FIG. 8).This result is consistent with the effect of p43 on the cultivatedfibroblasts. All of these results indicate that p43 can stimulate theproliferation of fibroblasts.

To further clarify that p43 stimulates the generation of endothelialcell and induces angiogenesis, the present inventors carried out severaldifferent experiments that can determine the activity of p43 inangiogenesis.

First, the present inventors investigated whether p43 induces themigration of endothelial cells by cellular wound migration assay. Inthis assay, the endothelial cell migration was enhanced in the presenceof p43 (See FIG. 9A). Then, the endothelial cells were cultivated onMatrigel and then the stimulated tube formation was also observed in thepresence of p43 (See FIG. 9B). Secondly, chorioallantoic membrane assay(CAM) was conducted. It could be seen that blood vessels were attractedto the area to which p43 was spotted (See FIG. 9C).

Lastly, the matrigel plugs containing p43 were used to see how p43affects neovascularization. As a positive control, the present inventorsused basic fibroblast growth factor (bFGF) which was known to beassociated with heparin in various forms and then to promoteangiogenesis, epithelization and the deposition of collagen fiber(Tsuboi, R. et al., J. Exp. Med. 172:245, 1990; Kinsnorth, A. N. et al.,Br. J. Surg. 77:409, 1990). The blood vessel formation within the gelwas monitored by immunostaining with the antibody specific toendothelial cells. It could be confirmed that the gels containing bFGFor p43 showed strongly enhanced neovascularization (See FIG. 10). Theresult of immunostaining with the antibody specific tomacrophage/monocyte was consistent with the result above (See FIG. 10).Further, it could be seen that the gels containing bFGF or p43 containedhigher concentration of red blood cells than the control (See FIGS. 10and 11).

BEST MODE FOR CARRYING OUT THE INVENTION

This invention is further illustrated by the following examples, whichare not be construed in any way as imposing limitations upon the scopethereof.

Example 1 Preparation of p43 Mutant Mouse

To prepare p43 mutant mice, p43 structural gene was allowed to bemutated using the gene trap method [Cecconi, F. & Meyer, B. I., FEBSLett., 480:63-71, 2000]. The gene trap vector, VICTR20 (LexiconGenetics, USA) was used to mutate the genomic DNA of SvEvBrd mice(Lexicon Genetics, USA) according to Zambrowicz's method [Zambrowicz, B.P. et al., Nature 392:608-611, 1998]. The mutated genomic DNA wasintroduced into the embryonic stem cells derived from 129/SvEvBrd mice(Omnibank) to generate the mutant library. In the library, the clonecontaining p43 gene disrupted by the insertion of the gene trap vectorwas screened and called ‘OST58507’. The clone was used to generate theheterozygous C57/BL6 mice (Samtako) by the standard method of LexiconGenetics. The mating of the heterozygous mice generated 16 of wild type,53 of heterozygous mutant mice and 28 of homozygous mutant mice. Theratio is close to the Mendelian segregation, indicating no significantembryonic lethality of the mutant.

Example 2 Characterization of p43 Mutant Mouse

2.1 Determination of the Gene Trap Vector's Site Inserted into p43Allele

To determine the site where the gene trap vector was inserted into p43allele, the p43 mutant allele was analyzed by sequencing. The sequencingwas determined by Pangenomcis, a company for sequence analysis. As shownin FIG. 1, the first exon (Exon I) and second exon (Exon II) of p43mutant gene were separated by the intron of about 7 kb. It was confirmedthat the gene trap vector was inserted at about I 0.5 kb downstream ofthe first exon.

2.2 Southern Blot

The genomic DNA was isolated from the tail of each mouse and digestedwith Ncol. The cleaved DNA fragments were separated by denaturing gelelectrophoresis. Then, southern blot analysis was performed using theneomycin resistant gene (Neo^(r))(SEQ ID NO: 2) of the gene trap vectoror the Hindu fragment (SEQ ID NO: 3) of the p43 mutant gene as a prove,according to a conventional method [Southern, E. M., J. Mol. Biol.,98:503, 1975]. As shown in FIG. 2A, the Neor gene of the gene trapvector and the p43 mutant gene (7 kb) were detected in the homozygousmutant mice. However, the Neo^(r) gene was not detected in the wild typemice. In the heterozygous mutant mice, the Neo^(r) gene was mildlydetected and both intact p43 gene (10 kb) and the p43 mutant gene (7 kb)were detected. The size of the p43 mutant gene (7 kb) was smaller thanthat of the intact p43 gene (10 kb) because of the presence of Nco Isite within Neo^(r) gene of the gene trap vector. From the results, itcan be demonstrated that the p43 mutant mouse of the present inventioncontains a mutation within the p43 gene.

2.3 PCR

To amplify the 1 kb DNA fragment containing the first intron of the p43gene, the genomic DNA was used as a template for PCR analysis with IF3primer (SEQ ID NO: 4) and IR3 primer (SEQ ID NO: 5) (See FIG. 1). Toamplify the 2.2 kb DNA fragment containing a portion of the gene trapvector and a portion of the p43 gene, pKOF2 primer (SEQ ID NO: 6) andthe IF3 primer were used in PCR analysis (See FIG. 1). For the PCRanalysis, the template DNA was denatured at a temperature of 94° C. for5 min and cycled 25 times at 94° C. for 1 min, at 57° C. for 1 min andat 72° C. for 1 min. As can be seen from FIG. 2B, only one band of 1 kbwas detected in the wild type mice (+1+) and two bands corresponding to1 kb and 2.2 kb, respectively, were detected in the heterozygous mutantmice (+/−). In the homozygous mutant mice (−/−), a band of 2.2 kb wasonly detected. From the results, it can be demonstrated that the p43mutant mouse of the present invention contains a mutation within the p43gene.

Example 3 Expression of p43 by p43 Mutant Mouse 3.1 Northern Blot

For Northern analysis, total cellular RNA was prepared from mouseembryonic fibroblast (MEF) cells using RNeasy Midi Kit (QIAGEN)according to the manufacturer's instruction. The isolated RNA wasseparated on denaturing gel, and then transferred to the Hybond-N⁺membrane (Amersham). The RNAs on the membranes were hybridized with thespecific probes for p43 set forth in SEQ ID NO: 7. As shown in FIG. 3A,p43 mRNA was detected in the wild type (+/+) and the heterozygous mutantmice, but was not detected in the homozygous mutant mice (−/−). Itindicates that p43 is not expressed in p43 mutant mice (−/−) of thepresent invention.

3.2 Western Blot

For Western blot analysis, the protein was isolated from the mouse organaccording to Ziak et al. (Ziak M, et al., Biochem. Biophys. Res. Commun.280:363-367, 2001). Western blotting was carried out with anti-p43antibody as described previously [Park S. G., et al., J. Biol. Chem.274:16673-16676, 1999]. As shown in FIG. 3B, p43 protein was detected inthe wild type (+/+) and the heterozygous mutant mice, but was notdetected in the homozygous mutant mice (−/−). It was consistent with theresult of the Northern blot.

Example 4 Stimulatory Activity of p43 in Wound Healing 4.1 GrossAppearance

For gross appearance, the back skin and panniculus carnosus muscle of 7to 8 weeks old mice (wild type and homozygous mutant mice) were excisedaccording to Werner et al. [Werner, S., et al., Science 266:819-822,1994]. Firstly, the present inventor disinfected the back skin of micewith 70% ethanol, and shaved fur from the back under avertinanesthetization. Then, the circular excision wound of bilateral symmetryhaving a diameter of 0.5 cm was prepared using a circular punch of 1 cmon the back skin. The wounds were left uncovered without the drugtreatment or dressing. Meanwhile, p43 was prepared from E. coli asdescribed previously [Park S. G., et al., J. Biol. Chem.274:16673-16676, 1999]. 4 μg of the purified p43 was dissolved in 5 μlof PBS buffer containing 20% glycerol. Each group of the wounds in 25wild type mice was treated with either p43 (4 ug/wound) or the PBSbuffer with 20% glycerol alone (control), 4 times at 2 days interval.Also, the p43 mutant mice were left without the treatment of p43. Thewound closure was monitored daily using the Image-Pro Plus Software(Media Cybernetics).

As shown in FIG. 4A, the wounds in the wild type mice treated with p43(WT+p43) recovered at much faster rate compared to those in thehomozygous mutant mice (Ho) or the wild type mice which were not treatedwith p43 with the lapse of time. However, the wounds in the homozygousmutant mice (Ho) recovered at a much slower rate compared to those inthe wild type mice which were not treated with p43 even after much timehad passed. The wound closure was calculated as the percentage of theinitial wound area. As shown in FIG. 4B, the wound area of the wild typemice treated with p43 (WT+p43) was significantly decreased with thelapse of time.

4.2 Histochemical Analysis

5 day-aged wounds were isolated with 3 mm of adjacent normal tissue,fixed overnight in Bouin's solution (Sigma, HT10-1-32), and thenembedded in paraffin.

Paraformaldehyde-fixed paraffin sections (6 μm) of the wounds werestained by the Masson Trichome procedure (Lund, L. R., et al., EMBO J.18:4645-4656, 1999). Only littermates of the same sex were used fordirect histological comparison.

As shown in FIG. 5, the wild mice treated with p43 (WT+p43) wereconfirmed to indicate the increase of granulation tissue (purple sitesbelow the wound areas) and rapid re-epithelialization by the increase ofadvancing epithelial layer (arrows). However, the homozygous mutant mice(Ho) showed little increase of advancing epithelial layer in the wounds.

4.3 Induction of Macrophage/Monocyte and Endothelial Cells by p43

5 day-aged wounds were isolated with 3 mm of adjacent normal tissue. Theisolated wounds were fixed in 4% paraformaldehyde in PBS and then frozenin the OCT compound (Sakura). The tyro-sections of the wounds wereanalyzed by immune-fluorescence. To detect macrophage/monocyte andendothelial cells, the frozen sections were incubated in 1.7 μg/ml ofanti-mouse MOMA-2 antibody (Serotec) and 1.0 μg/ml of anti-CD31 antibody(Pharmingen) that are specific to the cells, respectively. Afterincubation for 1 hour with the primary antibodies, 1.0 μg/ml ofFITC-conjugated secondary antibody was added and further incubated for 1hour. The wounds were then counterstained with propidium iodide (1μg/ml), mounted on slides and investigated using confocalimmunofluorescence microscopy (μRadience; BioRad).

As shown in FIG. 6, the wound tissues of the wild type mice treated withp43 (WT-1-03) showed the enrichment of the macrophage/monocyte while thewound tissues of the homozygous mutant mice (Ho) showed fewer numbers ofthe macrophage/monocyte. Also, the immunostaining of the wounds with theantibody specific to endothelial cells showed that vascularization wasalso significantly enhanced in the p43-treated wounds while the reversewas the case in the p43 mutants (See FIG. 6).

4.4 Effect of p43 on the Proliferation of Fibroblasts 4.4.1 Effect ofp43 on the Cultivated Fibroblasts

To determine the effect of p43 on the proliferation of foreskinfibroblasts, the foreskin fibroblasts (American Type Culture Collection)were cultivated in the presence of the different amounts of p43 (1, 10,and 100 ng/ml) for 48 h, and then in fresh medium containing 1 uCitritium-labeled thymidine for 4 h. After washing and lysing the cellswith RIPA buffer, the amounts of the incorporated thymidine werequantified by liquid scintillation counting.

As shown in FIG. 7, the proliferation of fibroblasts was significantlyincreased in dose-dependent manner by p43.

4.4.2 Histological Analysis

To determine the effect of p43 on the proliferation of dermalfibroblasts in the cutaneous, wound area, the cutaneous wounds weregenerated on the back skin of 7 to 8 weeks old mice according to Werneret al. [Werner, S., et al., Science 266:819-822, 1994]. Meanwhile, p43was prepared from E. coli as described previously [Park S. G., et al.,J. Biol. Chem. 274:16673-16676, 1999]. 4 μg of the purified p43 wasdissolved in 5 μl of PBS buffer containing 20% glycerol. The wounds in25 wild type mice were treated with either p43 (4 ug/wound) or the PBSbuffer with 20% glycerol alone (control), twice in every other days. Themice were sacrificed in 3 days after wounding and the wound area wasisolated for histological analysis. The cross-section of the wound areawas stained using hematoxylin and eosin. The boxed region in the wound(see FIG. 8 Left) was analyzed by mmuno-fluorescence with the antibodyagainst Ki-67 that is the marker of the cell proliferation (Ben-Izhak,O. et al. Ki-67 antigen and PCNA proliferation markers preducts survivalin anorectal malignant melanoma. Histopathology 41, 519-525, 2002). 3day-aged wounds were isolated with 3 mm of adjacent normal tissue. Theisolated wounds were fixed in 4% paraformaldehyde in PBS and frozen inOCT compound. The frozen sections (6 μm) were equilibrated 3 times withPBS for 5 min. Non-specific interactions were blocked with PBScontaining 0.1% Tween 20, 1% non fat milk for 2 h at room temperatureand sections were incubated with 1/50 diluted anti-KI67 antibody(Santacruz) in PBST(PBS containing 0.1% Tween 20) for 2 hrs at 37° C.The sections were washed 2 times with PBST and subsequently incubatedfor 1 h at 37° C. with the FITC conjugated antibody. The sections werecounterstained with propium iodide (10 ug/ml) for 15 min and washed 3times with PBST for 5 min. Fluorescence signals were examined under theconfocal immunofluorescence microscopy (Radiance, BioRad).

As shown in FIG. 8, p43 can stimulated the proliferation of fibroblasts.

Example 5 Effect of p43 on Angiogenesis 5.1 Cellular Wound MigrationAssay

To investigate whether p43 induces the migration of endothelial cells,the cellular wound migration assay was performed. First, aorta wasexcised from the butchered cattle and washed by dipping in cold PBSbuffer containing 1% penicillin and 0.25 μg/ml fungizone. Then, bovineaortic endothelial cells (BAECs) were isolated from the surface of thetracheal aorta using a razor blade and incubated in DMEM containing 20%fetal bovine serum (FBS). The incubated cell cultures were wounded witha razor blade in 2 mm in width and the injury lines were marked. Then,the cultures were washed with serum-free medium and further incubated inDMEM with 1% serum, 1 mM thymidine and 1 nM p43. The cells were allowedto migrate for 16 hours and then rinsed. A control was not treated withp43. The cultures were fixed with absolute methanol (100%) and stainedwith Giemsa (Chameleon Chemical).

As shown in FIG. 9A, the endothelial cell migration was enhanced in thepresence of p43. From the result, it was demonstrated that p43stimulates the endothelial cell migration.

5.2 Blood Tube Formation

From the result of Example 5.1 described above, it was confirmed thatp43 induced the endothelial cell migration. And then, to investigatewhether p43 induces blood tube formation, BAECs (5×10⁵ cells) werecultivated on Matrigel (Becton Dickinson) in the presence of 1 nM of p43at 37 for 6 hours. A control was not treated with p43. Then, the changesof cell morphology were captured by phase contrast microscopy.

As shown in FIG. 9B, the stimulated tube formation was observed in thepresence of p43, but in the control. The result indicated that p43stimulates tube formation and it was consistent with the endothelialcell migration.

5.3 Chorioallantoic Membrane Assay

Fertilized chick eggs were incubated in the humidified egg breeder at37° C. On the third day of incubation, about 2 ml of egg albumin wasremoved by an 18-gauge hypodermic needle to detach the developingchorioallantoic membrane (CAM) from the shell. After the incubation foradditional 6 days, thermanox coverslips (Nunc) loaded with 0.1 μg of p43(dissolved in PBS buffer containing 20% glycerol) were placed on the CAMsurface and vascularization was observed. A control was not treated withp43. As shown in FIG. 9C, blood vessels were attracted to the area towhich p43 was spotted.

5.4 Matrigel Plug Assay

To see how p43 affects neovascularization, matrigel plugs containing p43were subcutaneously injected into the skin of mice and stained by MassonTrichome method, and then red blood cells within the gels were counted.First, the Matrigel (0.25 ml, Becton Dickinson) was supplemented withp43 (1 ng/ml) plus heparin (9 U/ml, Sigma). PBS buffer containing 20%glycerol was used as a negative control. And as a positive control, 143ng/ml of bFGF (R&D systems) plus heparin was used, in which bFGF wasknown as a growth factor to stimulate the blood tube formation byassociating with heparin in a various form. The mixtures weresubcutaneously injected into 7 weeks old C57BL6/J mice (Samtako) in adose of 0.25 ml/site. The mice were sacrificed 7 days after theinoculation and the gels were removed, fixed in 4% paraformaldehyde for24 hours. The gel plugs were embedded in the OCT compound (Sakura) andthen sliced into sections of 10 μm thick using Cyo-microtome (Zeiss).The sections were reacted with anti-CD31 antibodies (Pharmingen) andanti-MOMA2 antibodies (Serotec) that are specific to the endothelialcells and macrophage/monocyte, respectively, and then stained by MassonTrichome method [Lund, L. R., et al., EMBO J. 18:4645-4656, 1999]. As aresult, in the presence of p43, neovascularization was strongly enhanced(See FIG. 10, top row) and macrophage/monocyte were also recruited tothe gel compared to the control (See FIG. 10, middle row, greenfluorescence). Interestingly, other cells were also enriched in the gelstreated with p43 (red fluorescence).

The blood vessel formation within the gels was also monitored bycounting red blood cells. As shown in FIG. 10 (bottom row, arrowheads),the gels containing bFGF or p43 contained higher concentration of redblood cells than the control. Further, the concentration of hemoglobinwithin the gels was determined as described previously [Passaniti, A.,et al., Lab. Invest., 67:519-528, 1992]. As shown in FIG. 11, the gelscontaining bFGF or p43 contained higher concentration of hemoglobin thanthe control.

While this invention has been described in connection with what ispresently considered to be the most practical and preferred embodiment,it is to be understood that the invention is not limited to thedisclosed embodiment and the drawings, but, on the contrary, it isintended to cover various modifications and variations within the spiritand scope of the appended claims.

The entire disclosure of Korea Patent Application No. 2002-42858, filedon Jul. 22, 2002 including its specification, claims, drawings andsummary are incorporated herein by reference in its entirety.

1. A method for stimulating wound healing in a subject in need thereof,comprising: administering to a wound of the subject an effective amountfor stimulating wound healing of a composition, wherein the compositioncomprises a polypeptide selected from the group consisting of: (a) apolypeptide having an amino acid sequence set forth in SEQ ID NO: 1; (b)a polypeptide having at least 70% sequence homology with the polypeptideof (a).
 2. The method according to claim 1, wherein the compositionfurther comprises at least one selected from the group consisting ofantibiotics, antihistamines, anti-inflammatory drugs, anti-viral drugs,anti-fungal drugs and growth factors.
 3. The method according to claim1, wherein the composition further comprises a pharmaceuticallyacceptable carrier.
 4. The method according to claim 1, wherein thecomposition is formulated with any one form selected from the groupconsisting of a powder, liniment, gel, lotion, cream, ointment, paste,puff, aerosol and suppository.
 5. The method of claim 1, wherein saidwound is selected from the group consisting of burn, ulcer, trauma,post-surgical, post-childbirth, chronic wound and dermatitis.
 6. Themethod of claim 5, wherein said burn is selected from the groupconsisting of sunburn, chemical burn, radiation burn and thermal burn.7. The method of claim 5, wherein said ulcer is selected from the groupconsisting of pressure ulcer, plaster ulcer and decubitus ulcer.
 8. Themethod for claim 5, wherein said chronic wound is selected from thegroup consisting of bedsores, pressure sores, diabetes-related and poorcirculation-related.
 9. The method of claim 6, wherein said dermatitisis selected from the group of consisting of impetigo, intertrigo,folliculitis and eczema.